System And Apparatus For Porously-Encapsulated Magnetic-Nanoparticle Biosensors

ABSTRACT

A system for measuring analyte concentrations has porous-walled nanocontainers containing multiple magnetic nanoparticles, the magnetic nanoparticles coated with a selective binder that is analyte-responsive and binds a the analyte, an indicator substance releasable from the selective binder by the analyte, or an indicator substance cleavable by the analyte, apparatus for exposing the nanocontainers to a fluid potentially containing the analyte, and magnetic spectroscopy of Brownian motion sensing apparatus for detecting agglutination of the nanoparticles or binding of analyte to the nanoparticles. The system is used in a method comprising coating magnetic nanoparticles with a selective binder, encapsulating the magnetic nanoparticles in porous nanocontainers, exposing the nanocontainers to a fluid potentially containing analyte, using magnetic spectroscopy of Brownian motion sensing apparatus to detect agglutination or binding of the nanoparticles, and translating Brownian motion spectra to analyte concentrations.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 61/659,788 filed 14 Jun. 2012.

GOVERNMENT INTEREST

This invention was made with government support under 1U54CA151662-01awarded by the National Institutes of Health-National Cancer Institute.The government has certain rights in the invention.

FIELD

The present document describes biosensors fabricated from multiplebiosensing nanoparticles encapsulated in porous nanocontainers.

BACKGROUND

Proteins and other molecules such as nucleic acids often have activesites that are capable of binding compounds of interest, or analytes,with great specificity. Highly selective biosensors having a protein,such as an enzyme, antibody, aptamer or other molecule, capable ofselectively binding such analytes are known in the art. Some suchsensors make use of a chemical reaction catalyzed by the protein as partof providing a detectable signal. In other sensors, such as surfaceplasmon sensors, binding of the compound of interest to the proteincauses a physical change in a resonance that can be detected by suitableequipment.

It is known that small nanoparticles undergo a random motion induced byimpact with randomly moving molecules called Brownian motion. Brownianmotion can be detected and monitored with a technique called MagneticSpectroscopy of Nanoparticle Brownian Motion (MSB), described in anarticle published as A. M. Rauwerdink, J. B. Weaver, “Measurement ofMolecular Binding Using The Brownian Motion of Magnetic NanoparticleProbes” Applied Physics Letters 96, 033702 (2010) and on the web in Feb.1, 2010 issue of Virtual Journal of Biological Physics Research. Themethod also appears on the web athttp://engineering.dartmouth.edu/reu/documents/CharlieTsai_FinalReport.pdf,and for which a copy is attached as an appendix hereto, the contents ofwhich are incorporated herein by reference. It is also known thatBrownian motion is a function of particle size, with larger, heavier,particles exhibiting smaller displacements than smaller, lighter,particles.

Magnetic nanoparticles, which typically have cores of either iron oriron oxide, have been coated with proteins or other molecules capable ofselectively binding to analytes. When such particles are in suspension,a change in Brownian motion as measured by MSB can be detected when theparticles are exposed to the analytes. Strong signal changes occur whenmultiple nanoparticles bind to the same analyte molecules and thereforeagglutinate or aggregate—agglutinated nanoparticles effectively formingfewer but larger and heavier nanoparticles in the suspension; similarsignal changes occur when the analyte binds nanoparticles to largerstructures such large beads or the solid surfaces. However, changes alsooccur when individual molecules of analyte bind individualnanoparticles.

SUMMARY

A system for measuring an analyte concentration in a solution has aplurality of nanocontainers suspended in the solution, eachnanocontainer having porous walls, at least some nanocontainerscontaining a plurality of magnetic nanoparticles, the magneticnanoparticles being coated with a selective binder such as a protein,aptamer, or other molecule capable of selectively binding the analyte,an indicator substance releasable from the selective binder by theanalyte, or an indicator substance cleavable by the analyte; andmagnetic spectroscopy of Brownian motion sensing apparatus for detectingagglutination of the nanoparticles or another change in the rotationalfreedom, hemodynamic or magnetic properties of the nanoparticles.

An embodiment is a system for measuring analyte concentrations hasporous-walled nanocontainers containing multiple magnetic nanoparticles,the magnetic nanoparticles coated with a substance that selectivelybinds the analyte, apparatus for exposing the nanocontainers to a fluidpotentially containing the analyte, and magnetic spectroscopy ofBrownian motion sensing apparatus for detecting agglutination of thenanoparticles.

In another embodiment, a system is used in a method comprising coatingmagnetic nanoparticles with a selective binding substance, encapsulatingthe magnetic nanoparticles in porous nanocontainers, exposing thenanocontainers to a fluid, using magnetic spectroscopy of Brownianmotion sensing apparatus to detect agglutination or binding of thenanoparticles, and translating Brownian motion spectra to analyteconcentrations.

In another embodiment, the nanoparticles would be bound together or toother beads in the probe until the analyte either breaks them free as anenzymatic action on the linking molecules or the analyte could displacethe linking molecules through higher affinity bonds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration that illustrates multipleprotein-coated nanoparticles in a nanocontainer.

FIG. 2 is a schematic illustration that illustrates multipleprotein-coated nanoparticles in a nanocontainer with an analyte present.

FIG. 3 is a block diagram of an apparatus for interrogating thenanoparticles within the nanocontainers to determine concentration of ananalyte.

FIG. 4 is a flowchart of a method for measuring a concentration of ananalyte using coated nanoparticles bound in nanocontainers.

FIG. 5 is an illustration of the apparatus of FIG. 3 with added bias andsweep coils for localizing concentrations of nanoparticles in the

DETAILED DESCRIPTION OF THE EMBODIMENTS

Techniques for forming porous nanocontainers are known, and have beenpublished in PCT/US2006/028564, published as WO2007014113, the contentsof which are incorporated herein by reference. Alternatively othersmall, and potentially metabolizable, nanocontainers that are porous tothe analyte but may contain nanoparticles might be used instead of thenanocontainers described in PCT/US2006/028564. The nanocontainers can beany size where the size is selected for its function or method ofdelivery.

It is proposed that magnetic nanoparticles of a first size be coatedwith a selective binder, such as selective-binding proteins, aptamer, orother molecules capable of selectively binding an analyte, where theanalyte can pass through pores of a second size smaller than the firstsize. Further, multiples of these nanoparticles are then encapsulated ineach of multiple nanocontainers. The pore size of walls of eachnanocontainer is selected to be between the first and second sizes, suchthat the magnetic nanoparticles in each nanocontainer are containedwithin that nanocontainer. The selective binder is typically determinedindividually for each analyte with which the system is to be used.

Multiple nanocontainers 100 are shown in suspension in FIG. 1 withoutanalyte present. Each nanocontainer has multiple protein-coated, oraptamer-coated, nanoparticles 102 within it. Nanoparticles 102 areseparated from each other, moving by Brownian motion within theirassociated nanocontainers 100.

In the presence of analyte 104 particles or molecules (FIG. 2), analyteparticles 104 may diffuse through pores in nanocontainer walls to enterthe nanocontainers. Once within the nanocontainers, analyte particles104 may bind the selective-binding molecules adherent to eachnanoparticle; in some embodiments this binding is reversible and apercentage of nanoparticles bound is dependent on a concentration of theanalyte. At least four potential mechanisms to alter the Brownian motionexist: The analyte binding the nanoparticles increases their sizeslowing their rotational Brownian motion. Alternatively, multiplenanoparticles can bind one or more epitopes on the analyte allowing thenanoparticles to aggregate again impacting their Brownian motion. Yetanother alternative is for the regions on the analyte to bind both thenanoparticle and another larger nonmagnetic structure such as a bead orthe nanocontainer. Yet another alternative is for the analyte to cleaveor displace molecules binding the nanoparticles together or to anotherstructure allowing the nanoparticles more freedom again impacting theirBrownian motion. In all four cases the Brownian motion of thenanoparticle will be changed which will change the MSB signal allowingthe changes to be measured remotely.

The ability of the nanoparticles to rotate freely can be measured in asensitive way using Magnetic Spectroscopy of Brownian Motion (MSB). Oneimplementation of an apparatus for MSB measurement is shown 150. ThisMSB apparatus has an amplifier 152 coupled to drive a drive coil 154 forproviding an alternating magnetic field to nanoparticles 156 implantedor infused into subject 158. Pickup coil 160 and reverse-wound balancingcoil 162 sense a response induced in the magnetic field by nanoparticles156. The response is amplified by amplifier 164, digitized, and read byprocessor 166. The sensitivity of the system is achieved by measuringthe harmonics of the drive frequency allowing very small signals to bemeasured because of the lack of other signals at the harmonicfrequencies. In a typical embodiment, the frequency of alternatingcurrent applied to drive coil 154 is swept across many measuredfrequencies in a range of frequencies while responses are measured ateach measured frequency to obtain a spectrum of the responses.

In an embodiment, the nanocontainers are self-assembled nanocontainersprepared according to Barjor Gimi, et al, Cell encapsulation andoxygenation in nanoporous microcontainers or for a similar product,Biomed Microdevices. 2009 December; 11(6): 1205-1212, (Gimi) availableon the web athtt.://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829986/pdf/nihms178484.pdf.

The nanocontainers of Gimi and utilized in this embodiment have twodiscrete components,

-   -   1) a hollow cubic base that is loaded with cells, and    -   2) a lid that closes the nanocontainer after it is filled with        its magnetic nanoparticle payload.

The hollow cubic base comprises a 50 micron (μm) thick bottom face, andfour 200 μm thick sidewalls on which a 50 μm ‘female’ structure ispatterned to accommodate a corresponding ‘male’ structure formed in thelid. To create this cubic base, first a 50 82 m thick SU-8 2025(Microchem, Newton, Mass.) photoresist layer was spun on a Pyrex wafer.This SU-8 layer was patterned to form the bottom face of the hollowedcubic base. Next, 200 μm thick SU-8 2075 was spun, patterned and bakedto form the four side walls of the hollowed cubic base. This 200 μmthick SU-8 layer was not photodeveloped to ensure uniform thickness ofthe next layer, a 50 μm thick SU-8 2025 which was patterned to form thefemale structure.

The SU-8 lid comprises a 100 μm thick rectangle for structuralintegrity, and a 35 μm male structure that interfaces with the femalestructure in the cubic base. Recessed within the lid is a matrix ofcylindrical wells with a thin, semi-permeable nanoporous SU-8 membraneat the base of the wells. The thin membrane was devised to permit rapidand selective molecular transport. The lid fabrication was started withthe deposition of a 4,000 Å Chromium (Cr) adhesion layer by electronbeam evaporation. 2,000 Å thick Cr alignment marks were formed on top ofthe Cr coated wafer using a lift-off process. 350 nm thin SU-8 2000.5was then spun and patterned to form the thin membrane for the formationof the nanopore arrays. Next, 70 nanometer (nm) Polymethyl methacrylate(PMMA) A2 photoresist was spun on the patterned 350 nm thin membrane andbaked. The nanopore arrays were formed on the PMMA A2 layer by ElectronBeam Lithography (EBL) using a 30 kV applied voltage, a 120 pA currentand a 0.9 nC line dose. For each lid, 16 arrays of 1,680 nanopores, eachof 1 μm length, were formed. To protect this patterned PMMA A2 duringthe etch process, 200 Å thick aluminum (Al) was deposited on the top andside walls using oblique-angle metal deposition. The 350 nm thin SU-8membrane was etched by O2 plasma in reactive ion etch (RIE). The Alprotection layer was then removed with Al etchant and the PMMA A2 layerwas removed by acetone. This process resulted in the formation of verydeep nanopores while retaining a small feature size and uniform crosssection. Next, 100 μm thick SU-8 2075 was spun and patterned to form themain rectangular structure of the lid. 35 μum thick SU-8 2025 was thenspun and patterned to form male structure on the periphery. Finally, theSU-8 lids were released from the wafer substrate using Cr wet etchant.The fabrication sequence for the microcontainer or nanocontainer lid isshown in FIG. 3. These nanocontainers and lids of Gimi are thereforeformed of a polymer, in this embodiment of polymerized SU-8 photoresist.

Porous nanocontainers of other shapes may be fabricated, and porousnanocontainers may be fabricated from other materials, such as analginate, as appropriate for each application.

In a particular embodiment, alginate nanocontainers are prepared using acommercial coaxial air flow bead generator procured from NiscoEngineering AG, Switzerland (www.nischo.ch). The unit comprises anozzle, a coaxial air flow generator and a syringe pump for alginateinfusion. The coaxial air stream pulls droplets of alginate-nanoparticlesuspension from the nozzle and extrudes droplets of alginate mixed withnanoparticles into a gelling bath including CaCl₂, to form thecontainers. The nanocontainer is removed from the CaCl₂ solution whenthe nanocontainer wall is thick enough leaving the core gel. As the gelcore inside the nanocontainer dissolves, the nanoparticles in the gelare left trapped in the core forming the nanoparticle fillednanocontainer. The nanocontainer size is controlled by varying the airflow and rate of injection of the alginate solution into the nozzle. Amagnetic stirrer keeps the containers separated during gelling.Monodispersed alginate containers of the order of ˜500 mm can beprepared by this method. Smaller containers can be prepared using anaerodynamic assisted bead generator, where the nozzle is enclosed underhigher pressure using compressed air. As a result the diameter of thecontainers is compressed while exiting the orifice producing a smallercontainer size of about one tenth the size of the nozzle. Nanocontainersof the order of 10 nanometers can be prepared using a nozzle of about100 nanometers in this way.

In another embodiment, a CaCl₂ (Calcium Chloride) solution withsuspended nanoparticles in it is formed into droplets that are placed inan alginate solution to form the porous shell around thenanoparticles—the CaCl₂ hardening the alginate adjacent to the CaCl₂.Once the porous nanocontainer font's, remaining CaCl₂ diffuses out ofthe container leaving the nanoparticles inside.

For purposes of this document, nanoparticle means a magnetic particlehaving a largest dimension of between several nanometers and onemillimeter (1*10⁻⁸ to 1*10⁻³ meters) covalently or otherwise conjugatedwith a targeting molecule capable of binding an analyte or of beingcleaved by an analyte. Generally the binding or cleaving is veryspecific for the targeted analyte.

For purposes of this document, nanocontainer means a container having alargest dimension of between several nanometers and one millimeter(1*10⁻⁸ to 1*10⁻³ meters) and a volume of between 1*10⁻²⁴ to 1.0*10⁻⁶liters and capable of encapsulating one or more nanoparticles. Amidrange nanocontainer means a nanocontainer having a largest dimensionof between fifty microns and 0.15 millimeter (5*10⁻⁵ to 1.5*10⁻⁴ meters)and a volume of between 1.25*10⁻¹⁰ to 3.375*10⁻⁹ liters. To avoidinterference with MSB measurements of magnetic nanoparticles containedin the nanocontainers, it is preferable that the nanocontainers beformed from non-ferromagnetic materials such as, but not limited to,polymers and non-ferromagnetic metals.

In an embodiment, the nanoparticles are loaded into the nanocontainersby applying and closing the lids in a suspension of nanoparticles.Excess nanoparticles are then removed from the suspension by washing andfiltering.

In an alternative embodiment, the porous nanocontainers are alginatebeads containing the nanoparticles.

In other alternative embodiments, the nanocontainers or similarcontainers made of either the same or a different material that containsthe nanoparticles and protects them from the immune system.

In an embodiment for in-vivo use, the nanocontainers are exposed to afluid potentially containing the analyte by aspirating thenanocontainers into a syringe and injecting them subcutaneously into aliving organism, such as a mammal or human subject, at an injectionsite; thereby exposing the nanocontainers to bodily fluids of thesubject that potentially contain the analyte. In a particularembodiment, the nanocontainers are injected directly or indirectly intoa lesion such as a tumor, where they may be used to monitor levels ofanalyte in the tumor environment. The apparatus for performing magneticspectroscopy of Brownian motion (MSB) is then positioned adjacent to theinjection site and a response is measured. Since the nanocontainers mayremain embedded in the subject for some time, and ongoing monitoring ofconditions within patient or tumor can be desirable, the apparatus forperforming MSB may be removed, then re-positioned adjacent to theinjection site or tumor at a later time and a further response ismeasured. In an alternative embodiment, a small, portable, MSB apparatusis left in place over tumor or injection site to make repeatedmeasurements and send the information to a remote monitoring station.The MSB sensing apparatus detects agglutination or binding of thenanoparticles as a result of any analyte present in the fluid; acalibration table may be used to convert a measured MSB response to adetermined analyte concentration. In an alternative embodiment, a set ofcalibration equations estimating the concentration in terms of MSBresponse is used to determine an analyte concentration from the MSBresponse.

In an alternative embodiment, for in-vivo use, the nanocontainers aredelivered into the venous system or the capillaries or the smallarteries of a living mammal, which may be a human, via injection intothe blood stream from a catheter, syringe, or other mechanism Thenanocontainers are then transported by blood into small arterioleswithin a target organ or tumor of the mammal, where they lodge, and canthereupon be used to monitor levels of analyte within the target organor tumor. In a particular embodiment, the nanocontainers are coated withanticoagulant to minimize fibrous clot formation.

In an alternative embodiment, for ex-vivo or in-vitro use, thenanocontainers or nanoparticles are deposited into a well of amultiple-well test plate; in a particular embodiment the nanocontainersare adhered to a floor of the well. A solution, which in an embodimentis plasma or blood, in another embodiment is an aqueous or alcoholicextract, and in another embodiment is a sample of a fluid such asdrinking water, is added to the well such that the solution maypenetrate the container and be exposed to the nanoparticles, after whichMSB measurements are performed to measure an agglutination of thenanoparticles.

In an alternative embodiment, for ex-vivo or in-vitro use, thenanoparticles are deposited into a well of a multiple-well test plate. Asolution, which in an embodiment is plasma or blood, in anotherembodiment is an aqueous or alcoholic extract, and in another embodimentis a sample of a fluid such as drinking water, is added to the well suchthat the solution is exposed to the nanoparticles, after which MSBmeasurements are performed to measure an agglutination of thenanoparticles. In another particular embodiment the nanoparticles areadhered to a floor of the well by the analyte after which the MSBmeasurements are performed to measure the rotational freedom of thenanoparticles. In another particular embodiment the nanoparticles areadhered to a floor of the well and cleaved from the well by the analyteafter which the MSB measurements are performed to measure the rotationalfreedom of the nanoparticles.

In an embodiment, the nanoparticles are coated with a single type ofselectively-binding molecule that selectively binds the analyte. In aparticular embodiment the selectively-binding molecule is a protein,which in a particular embodiment is an antibody that selectively bindsthe analyte, or an enzyme, in another embodiment the selectively-bindingmolecule is an aptamer formed of deoxyribonucleic acid (DNA) orribonucleic acid (RNA) having a particular nucleotide sequence.

In an alternative embodiment, a first portion of the nanoparticles arecoated with a selectively-binding molecule that selectively binds afirst region of the analyte and another portion of the nanoparticles arecoated with a second selectively-binding molecule that selectively bindsa second region of the analyte, thereby allowing a single molecule ofanalyte to bind two nanoparticles together. In a particular embodiment,the analyte is thrombin, and the selectively-binding molecules bind toseparate sites on thrombin, and detection sensitivity of 3.5 picomolarhas been obtained.

In an alternative embodiment, the nanoparticles are coated with aselectively-binding molecule that selectively binds a first region ofthe analyte and beads or nanocontainers are coated with a secondselectively-binding molecule that selectively binds a second region ofthe analyte, thereby allowing a single molecule of analyte to bind ananoparticle to a bead or nanocontainer.

In another particular embodiment, nanoparticles within thenanocontainers are linked to each other or to another bead or thenanocontainer itself with an indicator substance that can be attacked byan analyte. For example, but not limitation, in a particular embodimentthe nanoparticles are bound with collagen 4. An enzyme MMP-9 is capableof cleaving the collagen-4 thereby separating the nanoparticles. Thenanoparticles are exposed to a solution containing MMP-9, and apercentage of nanoparticles that become unbound is measured by using MSBtechniques, thereby permitting quantification of MMP-9.

In a particular embodiment, the first portion and second portion ofnanoparticles are of uniform size. In an alternative particularembodiment, the first portion of nanoparticles are particles of a firstsize and the second portion of nanoparticles are particles of a secondsize.

In an alternative embodiment, the nanocontainers are adhered onto asurface of a well of a test plate; a fluid potentially containing theanalyte is then deposited into the well. The apparatus for performingMSB is then positioned adjacent to the well of the test plate, and aresponse is measured. The MSB sensing apparatus detects agglutination orbinding of the nanoparticles as a result of any analyte present in thefluid as a difference in a measured MSB response from an MSB response ofunbound, unagglutinated, nanoparticles; a calibration table may be usedby a processor of the MSB sensing apparatus to convert a measuredresponse to a determined analyte concentration.

In another alternative embodiment for ex-vivo or in-vitro use, thenanocontainers are adhered onto a surface of a test plate; a fluidpotentially containing the analyte is then flowed over the plate. Theapparatus for performing MSB is then positioned adjacent to the testplate, and an MSB response is measured; additional fluid may becontinually flowed past the test plate and measurement of the MSBresponse is repeated periodically until an MSB response that meets alarmcriteria is obtained, or until the test plate is determined to haveexpired. In this alternative embodiment, the apparatus may be used forscreening large quantities of fluid, such as domestic tap water, in afluid-handling apparatus for presence of a specific analyte, such as atoxin, chemical warfare agent, or other substance; when the analyte is,or becomes, present the MSB response meets the alarm criteria and analarm condition may be reported and/or an operating mode of thefluid-handling apparatus may be changed. The MSB sensing apparatusdetects agglutination or binding of the nanoparticles as a result of anyanalyte present in the fluid; a calibration table may be used to converta measured response to a determined analyte concentration. In aparticular embodiment, the nanoparticles are tagged with a synthetic DNAstrand complimentary (or countersense) to an analyte DNA strand.Experiments show that concentrations of the analyte DNA strand of onenanomolar or less can be detected and quantified by exposing thenanoparticles to a solution containing the analyte DNA in vivo or invitro. In a particular embodiment 12-pair nucleotide sequences are usedthat are complementary to a portion of an analyte DNA sequence.

In an embodiment using a streptavidin-biotin binding system, we found adetection limit of the MSB technique using our current apparatus islower than 150 picomolar.

A method for using nanocontainer-bound nanoparticles for sensing ananalyte is illustrated in FIG. 4. Magnetic nanoparticles are coated 302with a substance, such as a protein or a nucleic acid chain, thatselectively binds an analyte as previously discussed. Nanocontainers arethen prepared 304. The nanoparticles are then encapsulated 306 into thenanocontainers. The nanocontainers are then exposed 308 to a solutionthat potentially may contain the analyte, which in some embodiments isdone by injecting nanocontainers into a living mammal or human subject,and in other embodiments is done by binding nanocontainers on a plateand adding solution. The MSB response of the nanoparticles is thenmeasured 310. Measured MSB response is used 312 to find an analyteconcentration by using a calibration chart.

While most of the foregoing discussion references nanoparticles in thenanocontainers becoming bound when exposed to analyte, or released by anenzymatic reaction, in another embodiment, nanoparticles tagged with aselective binding aptamer or protein (including antibodies) are bound byexposure to an indicator substance that includes a linking moleculehaving multiple binding sites resembling a binding site of an analyte.The nanocontainers and nanoparticles may then be desiccated and stored,then rehydrated, and may be injected into a subject, placed in atest-plate well, or bound to a test plate. The nanoparticles are thenexposed to a solution that may contain analyte. In this embodimentexposure of nanoparticles to the analyte displaces the linkingindicator-substance molecules through competitive binding or higheraffinity bonding of analyte to the selective binding aptamer or protein,thereby releasing agglutinated nanoparticles from each other, or fromnanocontainer walls. Releasing agglutinated nanoparticles from theirpre-analyte agglutinated or aggregated state causes a detectable changein MSB response that can be quantified and used to determine analyteexposure or analyte concentration.

In another embodiment, with reference to FIG. 5, bias-field 190 andcomplementary sweep 192 magnets are added to the MSB detectionapparatus. If the bias-field magnet is strong enough to saturate themagnetic nanoparticles—drowning out MSB measurement, but adjustablesweep magnets are strong enough to create a zone of near-zero bias fieldwhere an MSB response can be measured, the zone of near-zero field canbe positioned at will within a subject. MSB signal from encapsulatednanoparticles in the near-zero field position can be determined, whileMSB signals from elsewhere in the subject can be excluded from themeasurement. By changing field in sweep magnets, the zone of near-zerofield can be moved through the subject, allowing MSB signals to bedetermined, and both magnetic nanoparticle concentration and analyteconcentration determined, at multiple positions within the subject.These multiple concentrations are then mapped and displayed as atomographic image.

Combinations

The herein-described nanoporous nanocontainers containing magneticparticles, and their use to measure an analyte, may be used in a varietyof ways with a variety of analytes. In particular, some of thecombinations are as follows.

A system designated A for measuring an analyte concentration in asolution has multiple nanocontainers each having porous walls, at leastsome nanocontainers containing a plurality of magnetic nanoparticles,the magnetic nanoparticles coated with a selective binder capable ofselectively binding a substance selected from the group consisting ofthe analyte, and an indicator substance releasable from the selectivebinder by the analyte, and an indicator substance cleavable by theanalyte, apparatus for exposing the nanocontainers to the solution, andmagnetic spectroscopy of Brownian motion sensing apparatus for detectingagglutination or aggregation of the nanoparticles.

A system designated AA including the system designated A wherein theselective binder is selected from the group consisting of a nucleic acidstrand, a protein, and a combination of nucleic acid strands andprotein.

A system designated AB including the system designated A or AA whereinthe nanocontainers are of less than one millimeter in largest dimension.

A system designated AC including the system designated AB, A or AAwherein the nanocontainers are midsize nanocontainers having averagesize less than 150 microns in largest dimension.

A system designated AD including the system designated A, AA, AB, or ACwherein the nanocontainers are formed of photoresist.

A system designated AE including the system designated A, AA, AB, or ACwhere the nanocontainers are formed of an alginate.

A system designated AF including the system designated A, AA, AB, AC,AD, or AE where the apparatus for exposing the nanocontainers to thesolution comprises apparatus for injecting the nanocontainers into aliving mammal.

A system designated AG including the system designated A, AA, AB, AC,AD, AE, or AF where the apparatus for exposing the nanocontainers to thesolution comprises a test plate to which nanocontainers are adhered.

A system designated AGA including the system designated A, AA, AB, AC,aD, AE, or AF, where the apparatus for exposing the nanoparticles to thesolution comprises a well of a multiple-test plate within whichnanocontainers with nanoparticles are deposited.

A system designated AH including the system designated A, AA, AB, AC,AD, AE, AF, AG, or AGA where the selective binder is a nucleic-acidchain.

A system designated AI including the system designated A, AA, AB, AC,AD, AE, AF, AG, or AGA where the selective binder is a protein.

A system designated AJ including the system designated A, AA, AB, AC,AD, AE, AF, AG, or AGA where the selective binder is an antibody.

A system designated AK including the system designated A, AA, AB, AC,AD, AE, AF, AG, AGA, or AK, wherein the selective binder is configuredto bind analyte.

A system designated AL including the system designated A, AA, AB, AC,AD, AE, AF, AG, AGA, or AK, further comprising an indicator substanceand wherein the selective binder is configured to bind the indicatorsubstance in such a way that the analyte can displace indicatorsubstance from the selective binder.

A system designated AM including the system designated A, AA, AB, AC,AD, AE, AF, AG, AGA, or AK, further comprising an indicator substanceand wherein the indicator substance is cleavable by the analyte.

A method designated B of determining a concentration of an analyte in afluid including coating a plurality of magnetic nanoparticles with aselective binder selected from the group consisting of a protein and ora nucleic acid and capable of binding a substance selected from thegroup consisting of the analyte, and an indicator substance releasablefrom the selective binder by the analyte, and an indicator substancecleavable by the analyte; encapsulating the magnetic nanoparticles inporous nanocontainers; exposing the nanocontainers to the fluid; usingmagnetic spectroscopy of Brownian motion sensing apparatus to determinean MSB response dependent upon agglutination or binding of thenanoparticles.

A method designated BA including the method designated B wherein thestep of using magnetic spectroscopy of Brownian motion sensing apparatusto determine an MSB response is repeated over a period of time.

A method designated BB including the method designated BA furtherincluding determining an alarm condition based upon the MSB response.

A method designated BC including the method designated B, BA, or BBfurther comprising injecting the porous nanocontainers into a livingmammal, and wherein the fluid is a bodily fluid of the living organism.

A method designated BD including the method designated B, BA, BB or BCwherein the nanocontainers are of less than one millimeter in largestdimension.

A method designated BE including the method designated BD wherein thenanocontainers are midsize nanocontainers having average size less than150 microns in largest dimension.

A method designated BF including the method designated B, BA, BB, BC,BD, or BE where the nanocontainers are formed of polymer.

A method designated BG including the method designated B, BA, BB, BC,BD, or BE where the nanocontainers are formed of an alginate.

A system designated C for measuring an analyte concentration a pluralityof magnetic nanoparticles, the magnetic nanoparticles coated with aselective binder capable of selectively binding a substance selectedfrom the group consisting of the analyte, an indicator substancereleasable from the selective binder by the analyte, and an indicatorsubstance cleavable by the analyte, apparatus for exposing thenanocontainers to the solution, and magnetic spectroscopy of Brownianmotion sensing apparatus for detecting agglutination or aggregation ofthe nanoparticles.

A system designated CA including the system designated C, wherein theselective binder binds an indicator substance.

A system designated CB including the system designated CA or C, wherethe apparatus for exposing the nanoparticles to the solution comprises awell of a multiple-test plate within which nanocontainers withnanoparticles are deposited.

A system designated CC including the system designated CA, C, or CBwhere the selective binder is a nucleic-acid chain.

A system designated CD including the system designated C, CA, or CBwhere the selective binder is a protein.

A system designated CE including the system designated CD where theselective binder is an antibody.

A system designated CF including the system designated C, CA, CB, CC, CDor CE, wherein the selective binder is configured to bind analyte andwherein the selective binder is configured to bind the indicatorsubstance in such a way that the analyte can displace indicatorsubstance from the selective binder.

A system designated CG including the system designated C, CA, CB, CC,CD, or CE, wherein the indicator substance is cleavable by the analyte.

While the invention has been particularly shown and described withreference to a preferred embodiment thereof, it will be understood bythose skilled in the art that various other changes in the form anddetails may be made without departing from the spirit and scope of theinvention. It is to be understood that various changes may be made inadapting the invention to different embodiments without departing fromthe broader inventive concepts disclosed herein and comprehended by theclaims that follow.

1. A system for measuring an analyte concentration in a solutioncomprising: a plurality of nanocontainers having porous walls, at leastsome nanocontainers containing a plurality of magnetic nanoparticles,the magnetic nanoparticles coated with a selective binder capable ofselectively binding a substance selected from the group consisting ofthe analyte, an indicator substance releasable from the selective binderby the analyte, and an indicator substance cleavable by the analyte;apparatus for exposing the nanocontainers to the solution; and magneticspectroscopy of Brownian motion sensing apparatus for detectingagglutination or aggregation of the nanoparticles.
 2. The system ofclaim 1 wherein the selective binder capable of selectively binding theanalyte is selected from the group consisting of a nucleic acid strand,a protein, and a combination of nucleic acid strands and protein.
 3. Thesystem of claim 2 wherein the nanocontainers are of less than onemillimeter in largest dimension.
 4. The system of claim 3 wherein thenanocontainers are nanocontainers having average size less than 150microns in largest dimension.
 5. The system of claim 4 where thenanocontainers are formed of photoresist.
 6. The system of claim 4 wherethe nanocontainers are formed of an alginate.
 7. The system of claim 3,where the apparatus for exposing the nanocontainers to the solutioncomprises apparatus for injecting the nanocontainers into a livingmammal
 8. The system of claim 3, where the apparatus for exposing thenanocontainers to the solution comprises a test plate to whichnanocontainers are adhered.
 9. The system of claim 3, where theselective binder capable of selectively binding the analyte is anucleic-acid chain.
 10. The system of claim 3, where the selectivebinder capable of selectively binding the analyte is a protein.
 11. Thesystem of claim 10 where the selective binder capable of selectivelybinding the analyte is an antibody.
 12. The system of claim 3, where theapparatus for exposing the nanoparticles to the solution comprises awell of a multiple-test plate within which nanoparticles are deposited.13. The system of claim 3, wherein the selective binder is configured tobind analyte.
 14. The system of claim 3, further comprising an indicatorsubstance and wherein the selective binder is configured to bind theindicator substance in such a way that analyte can displace indicatorsubstance from the selective binder.
 15. The system of claim 3, furthercomprising an indicator substance and wherein the indicator substance iscleveable by the analyte.
 16. A method of determining a concentration ofan analyte in a fluid comprising: coating a plurality of magneticnanoparticles with a selective binder selected from the group consistingof a protein and or a nucleic acid and capable of binding a substanceselected from the group consisting of the analyte, an indicatorsubstance releasable from the selective binder by the analyte, and anindicator substance cleavable by the analyte; encapsulating the magneticnanoparticles in porous nanocontainers; exposing the nanocontainers tothe fluid; using magnetic spectroscopy of Brownian motion sensingapparatus to determine an MSB response dependent upon agglutination orbinding of the nanoparticles.
 17. The method of claim 16 wherein thestep of using magnetic spectroscopy of Brownian motion sensing apparatusto determine an MSB response is repeated over a period of time.
 18. Themethod of claim 17 further comprising determining an alarm conditionbased upon the MSB response.
 19. The method of claim 18 furthercomprising injecting the porous nanocontainers into a living mammal, andwherein the fluid is a bodily fluid of the living organism.
 20. Themethod of claim 16 wherein the nanocontainers are of less than onemillimeter in largest dimension.
 21. The method of claim 20 wherein thenanocontainers are midsize nanocontainers having average size less than150 microns in largest dimension.
 22. The method of claim 19, where thenanocontainers are formed of polymer.
 23. The method of claim 19, wherethe nanocontainers are formed of an alginate.
 24. (canceled)
 25. Themethod of claim 19, wherein the selective binder is configured to bindanalyte.
 26. The method of claim 19, wherein the selective binder isconfigured to bind the indicator substance in such a way that analytecan displace indicator substance from the selective binder, and furthercomprising binding nanoparticles with the indicator substance.
 27. Themethod of claim 19 wherein the selective binder is configured to bindthe indicator substance, and wherein the indicator substance iscleavable by the analyte.
 28. The method of claim 28 wherein theselective binder is configured to bind an indicator substance, andwherein the indicator substance is bound to the nanocontainer.
 29. Asystem for measuring an analyte concentration comprising: a plurality ofmagnetic nanoparticles, the magnetic nanoparticles coated with aselective binder capable of selectively binding a substance selectedfrom the group consisting of the analyte, an indicator substancereleasable from the selective binder by the analyte, and an indicatorsubstance cleavable by the analyte, wherein the magnetic nanoparticlesare contained within nanocontainers; apparatus for exposing thenanocontainers to the solution, and magnetic spectroscopy of Brownianmotion sensing apparatus for detecting agglutination or aggregation ofthe nanoparticles.
 30. A system comprising the system of claim 29,wherein the selective binder binds an indicator substance.
 31. A systemcomprising the system of claim 30 where the apparatus for exposing thenanoparticles to the solution comprises a well of a multiple-test platewithin which the nanocontainers are bound.
 32. A system comprising thesystem of claim 31 where the selective binder is a nucleic-acid chain.33. A system designated comprising the system of claim 31 where theselective binder is a protein.
 34. A system comprising the system ofclaim 33 where the selective binder is an antibody.
 35. A systemcomprising the system of claim 31 wherein the selective binder isconfigured to bind analyte and wherein the selective binder isconfigured to bind the indicator substance in such a way that theanalyte can displace indicator substance from the selective binder. 36.A system comprising the system of claim 31, wherein the indicatorsubstance is cleavable by the analyte.